(-)-Huperzine A (1), a Lycopodium alkaloid isolated from Chinese folk medicine Huperzia serrata, has attracted a great deal of interests as a new drug for the treatment of Alzheimer's disease (AD) due to its potent, reversible and selective inhibitory activity against acetylcholinesterase (AChE). Because of the interesting biological activity coupled with the unique structural features, a bicyclo[3.3.1] skeleton and pyridone moiety, we initiated our efforts on the total synthesis of this fascinating molecule. Herein, we describe a total synthesis of huperzine A (1) featuring a unique cation-olefin cyclization. Our synthesis commenced with a construction of the bicyclo[3.3.1] core skeleton. Diels-Alder reaction between furan and maleic anhydride provided anhydride 9. Reduction of the anhydride followed by acidic work-up gave the lactone, which was subjected to hydrogenation to furnish lactone 6. The chemo-, regio-, and stereoselective alkylation of 6 was accomplished by treatment with two equivalents of KHMDS to generate dianion 10, which was alkylated with methallyl bromide to give homoallyl alcohol 5. A vanadium-catalyzed epoxidation of 5 gave epoxyalcohol 11, which was converted into α, β-unsaturated ketone 4 by means of Swern oxidation. Upon treatment with TBSOTf in the presence of base, 4 underwent unexpected cyclization to give silyl enol ether with bicyclo[3.3.1] skeleton, which was desilylated to provide ketone 13. More conveniently, ketone 13 was obtained by treatment of 4 with a catalytic amount of TIPSOTf in one step. We next focused on introduction of the amino functionality. After protection of the secondary alcohol with a MOM group, the lactone in 14 was opened by treatment with 1 equivalent of PhSK. The resulting carboxylic acid 15 was converted to methyl carbamate 17 via Curtius rearrangement. Subsequent treatment with mCPBA followed by β-elimination of sulfoxide provided the desired α, β-unsaturated ketone 19. Construction of the pyridone moiety was performed via a pyrone. After Michael reaction of sulfinyl amide to 19, ketoamide 22 was converted to pyrone 23 in refluxing toluene. Treatment of 23 with aqueous NH_3 under reflux gave pyridone 24 which was protected as its 2-methoxypyridine 25. Toward a total synthesis of huperzine A (1), we investigated the introduction of the ethylidene moiety. After cleavage of the MOM group, the resulting alcohol was oxidized by Swern oxidation. Since Wittig reaction is known to give the wrong stereochemistry, ketone 26 was converted into allyl chloride 28 via a two-step procedure including addition of vinyllithium and treatment with SOCl_2. Reduction of allylic chloride with LiBHEt_3, followed by demethylation with TMSI afforded huperzine A (1). Optically active lactone 6 could be obtained via an asymmetric opening of anhydride 9 using quinine according to the Bolm's procedure. Synthesis of (-)-huperzine A (1) is currently under way.

抄録全体を表示

(+)-Pestalotiopsin A (1) was isolated from an endophytic fungus of the Pacific yew tree and showed cytotoxicity and immunosuppressive activity. The structure of 1 is comprised of a cyclobutane ring fused with both an (E)-cyclononene ring and a γ-lactol unit. So far three groups, in addition to our group, have reported a number of synthetic studies of 1. Herein, we report the first total synthesis of (-)-pestalotiopsin A (2), thereby establishing the absolute stereochemistry of natural 1. The total synthesis began with the [2+2] cycloaddition of N-propioloyl Oppolzer's camphorsultam 6 and ketene acetal 7. The 1,4-hydride addition to the adduct 8 and subsequent protonation provided the cyclobutane derivative 9 with an excellent level of stereoselection. Removal of the camphorsultam from 9 provided cyclobutanemethanol 10 with >95% ee, which was converted into the bicyclic lactone 4 in 8 steps. The aldol reaction of 4 with the chiral aldehyde 5, prepared from the known epoxy chloride 16, provided the desired anti-aldol product 21 predominantly. The intramolecular NHK reaction of aldehyde/alkenyl iodide 3, which in turn was derived from 21, proceeded smoothly to provide the cyclized product 23 in 92% yield as a single diastereomer. Considering the highly strained nature of the (E)-cyclononene moiety, the observed high yield of 23 was remarkable. After some examinations examined for the removal of the hydroxy group introduced in 23, we eventually found that the palladium(0)-catalyzed reduction of the mesylate 27 in the presence of NaBH_4 proceeded well without isomerization of the (E)-olefin moiety into the Z-form, providing 28. Finally, the deoxygenated product 28 was converted into (-)-pestalotiopsin A (2), the antipode of natural (+)-pestalotiopsin A (1), in 3 steps.

抄録全体を表示

Many natural stilbenoids in Dipterocarpaceae and Vitaceae exist as 'oligomer' in which some stilbenoid molecules are coupled with other at various positions. These oligomeric stilbenoids have been shown to possess a wide range of physiological and biological properties including anticancer, anti-inflammatory, and antiviral activities. The oligomeric stilbenoids in Dipterocarpaceaeous plants have been our main focus of extensive structural investigation for the past decade. Although a detailed structural determination based on NMR spectra is required as a part of ongoing chemical investigation, the difficulties caused by complicated stereochemistry that comprises diastereomer, epimer and enantiomers make the structural determination somewhat difficult to tackle. In the present study, the structural characterization of shoreaketone (1) isolated from three Dipterocarpaceaeous plants is described, because 1 appears different from other resveratrol tetramers with respect to its skeleton and NMR spectral complexity. The heterocyclic ring in 1 is unique and has not been recorded in any other natural product that contains a dense array of functionality and stereochemistry. We have identified a resveratrol tetramer with a chiral axis in 1 for the first time. Our findings demonstrate complex chemical and dynamic behavior in 1. From detailed spectral data analysis to examine the mutual effect of the hindered rotation around the chiral axis, we could demonstrate the presence of two rotamers (1a and 1b). Using edited ROESY, NOESY, and differential NOE experiments, we propose that both rotamers exhibit rotational conformation exchanges. The formation of the symmetrical molecule, isohopeaphenol (2) solves the stereochemical aspect and stereostructure could be confirmed by demethylation after the acid catalyzed skeletal transformation of 1. From theoretical calculations, the barrier to rotation of the chiral axis was found to be around 33.7kcal/mol. Our study signifies a new dimension toward the ongoing research in this field by exploring such type of complex systems bearing a 2,3-diaryl-dihydrobenzofuran ring. The structure of isohopeaphenol (2) was confirmed by spectroscopic evidences and skeletal conversion of 1.

抄録全体を表示

A novel γ-methylidene-spirobutanolide, spirolephtoshol (1) and it's biosynthetic congeners leptospholic acid (7), spiroleptosphol B (8), spiroleptosphol C (10) and norleptosphol C (11) were isolated from ascomycetous fungus Leptosheria doliolum. Biological assay revealed that 1 exhibits cyctotoxicity against P388 marine leukemia (EC_<50>=20μg/mL) and HeLa human cervix carcinoma (EC_<50>=5μg/mL). The relative structure of 1 was established by NMR analysis involving NOE experiments. The absolute structure of the bicyclic moiety was determined by chemical dericatization followed by the CD analysis. The relative and absolute stereochemistry of the side chain was revealed by spectral and chiral GC analysis of the degradation product with synthetic samples. In spite of broad ^1H NMR spectra, 7 was established to be a mixture of 7a and 7b by determing the corresponding acatets. The extend ^1H and ^<13>C NMR analyses disclosed the structures of 8, 10 and 11. Biosynthetic studies based on ^<13>C labeling profiles, suggest 1 is consist of heptaketide with two methyl groups derived from methionine. Now we propose that C3 unit of the spirorinn moiety(C4, C3, C11) is also derived from acetates but after conversion in the TCA cycle. Detailed biological propaties and the biosynthesis of these compounds are now under investigation in our laboratories.

抄録全体を表示

Intra-specific diversity in L. tsangchanensis, L. latihastata, L. lankongensis, L. duciformis, and their related species growing in Yunnan and Sichuan provinces of China was studied by chemical and genetic approaches. L. tsangchanensis samples collected in Yunnan and in Sichuan could be distinguished based on the root constituents and the base sequences of the internal transcribed spacers (ITSs) of the ribosomal RNA gene. The Yunnan samples were found to contain cacalol (1) as the major component, while samples from Sichuan contained eremophila-9, 11-dien-8-ones (5,6) and their derivative as the major components. Intra-specific diversity in L. latihastata was found to be small in chemical composition and large in the atpB-rbcL sequence. In all collected L. latihastata and L. villosa samples, two major components, 2-isopropenyl-5, 6-dimethoxybenzofuran (11) and 2-isopropenyl-5-acetyl-6-methoxybenzofuran (euparin; 12) were detected, indicating that the two species are chemically close to each other. Five highly oxygenated bisabolane-type sesquiterpenes were isolated from L. lankongensis, including four new compounds 15, 17-19. A small intra-specific diversity was observed in the composition of the root chemicals but not in the DNA sequence. L. duciformis collected in Sichuan was found to contain sinapyl or coniferyl alcohols (20-23), while the species in Yunnan contained oplopanes (24,25) or eremophilane sesquiterpenes (8,9). Diversity was observed in the atpB-rbcL sequence as well.

抄録全体を表示

Seven triterpenoid glycosides and two phenylpropanoid glycosides were isolated from the fruits of Ilex rotunda. These compounds were determined based on UV, MS, IR ^1H-NMR, ^<13>C-NMR and 2D-NMR spectroscopic data as 3-O-β-glucuronopyranosyl oleanolic acid [1], 3-O-β-glucuronopyranosyl-olean-12-ene-28-oic acid 28-O-β-glucopyranoside [2], 3-O-β-glucuronopyranosyl-23-hydroxyl methyl olean-12-ene-28-oic acid [3], 23-hydroxyl methyl olean-12-ene-28-oic acid [4], 3-O-β-glucuropyranosyl-19-O-β-glucopyranosyl-23-hydroxyl methyl olean-12-ene-28-oic acid [5], 3-O-β-glucuronopyranosyl-19-hydroxy-23-hydroxyl methyl olean- 12-ene-28-oic acid 28-O-β-glucopyranoside [6], 19-hydroxy-olean 12-ene-28-oic acid [7], syringin [8] and chlorogenic acid [9]. And we examined their superoxide scavenging activity and tyrosinase inhibitory activity (Step 1 as L-Tyrosine substrate; Step 2 as L-DOPA substrate), and their compounds investigated for cosmetic use. As a result, for superoxide scavenging activity, the 1-butanol extract and aqueous phase part were good activity of 69.30 and 72.22%, respectively. For tyrosinase inhibitory activity, the 1-butanol extract, good inhibition of 45.05% of Step 1 (L-Tyrosine substrate) was noted. Melanin inhibition and cytotoxicity were examined by using cultured B16 melanoma cells. More than 60% melanin inhibition was observed in the 1-Hexan extract, and methanol eluate of an Amberlite XAD-2. In addition, remarkable melanin inhibitions were found in [1] and [5] which are those isolation compounds. Among these isolated compounds, [5] is a new triterpenoid.

抄録全体を表示

The red alga in genus Gracilaria are known to contain prostaglandins which were suspected to be the causative agents of lethal human intoxication caused by ingestion of these red alga in Japan. As the results of our effort to find novel prostanoids and eicosanoids, we isolated a new prostanoid named UK365 from G. edulis collected in Philippines, and two new hydroxy eicosanoids from Japanese G. vermiculophylla. The planar structures of these three new compounds were determined by 2D NMR techniques. UK365 was suggested to contain a γ-hydroxybutenolide moiety in the molecule. Two new eicosanoids from G. vermiculophylla were determined as 4,8-dihydroxyeicosa-(5Z,9E,11Z,14Z)-tetraenoic acid (4,8-diHETE) and 4,8,15-trihydroxyeicosa-(5Z,9E,11Z,13E)-tetraenoic acid (4,8,15-triHETE).

抄録全体を表示

Cathepsins, collectively known as a class of lysosomal proteindegrading enzymes, have been shown to possess specific physiological activities including cancer progression. The contribution of cathepsin B in tumor invasion is well documented and the inhibition of its activity resulted in a decreased invasiveness of tumor cells. Therefore, the inhibitors of cathepsin B have a potential as anticancer agents. In our search for anticancer lead compounds from Japanese marine invertebrates, we found activity in the extract of the marine sponge Crella (Yvesia) spinulata. Bioassay-guided fractionation afforded two new sterol dimers named shishicrellastatins A (1) and B (2). The structure of shishicrellastatin A (1) was elucidated by interpretation of spectral data. Relative stereochemistry was elucidated by analysis of the ROESY spectrum. Shishicrellastatin B (2) was a dehydrogenated derivative of 1. Compounds 1 and 2 exhibited cathepsin B inhibitory activity.

抄録全体を表示

The importance of the endoplasmic reticulum (ER) in triggering a specific program of cell death has been recently reported. By triggering apoptosis on neural cells, ER-stress is a major cause of neurodegenerating disease such as Alzheimer disease. The demand for new protective substances to the ER-stress-dependent cell death prompted us to screen the protective activity of mushrooms. On the course of the screening, we isolated new hericenones, 3-hydroxy-hericenone F as a protective agent, along with related compounds, hericenone I and J. The structures of hericenones were determined by NMR and MS spectra. The structure of 3-hydroxy hericenone was elucidated as (8-formyl-3-hydroxy-5-methoxy-2-methyl-2-(4'-methyl-2'-oxopent-3'-enyl) chroman-7-yl) methyl palmitate, which had one additional hydroxy group compared to hericenone F. 3-Hydroxy-hericenone F showed the protective activity dose-dependently, however hericenones F, I and J did not have any activity at the concentration of 10μg/mL. Therefore, we suggested that the hydroxy group of 3-hydroxy-hericenone F was important for the protective activity. This is the first report of a new hericenone which has the protective activity against ER stress-dependent cell death. The further study using more hericenones is needed for the understanding of the structure-activity relationship.

抄録全体を表示

The peach (Prunus persica) is a species of Prunus native to China that bears an edible juicy fruit. We investigated the bioactive function of extracts of peach leaf to achieve a more effective use of resources. In the present study, we found that the extract of peach leaf showed anti-hyperglycemic activity in mice. It may thus be useful for preventing the postprandial absorption of carbohydrates used as drugs to treat diabetic patients and as functional food for people who need to control their own blood glucose level. The methanol extract of peach leaf suppressed the postprandial blood glucose level after an oral administration of soluble starch, maltose, sucrose and glucose in mice. The mechanism of action is proposed to be due to the separate components, the carbohydrate digestive enzyme-inhibiting activity and the glucose absorption-inhibiting activity in intestine, based on the results of in vitro and in vivo experiments. To clarify the mechanism of inhibitory activity on postprandial blood glucose elevation, the inhibitory effect of fractionated components from the crude extract on the glucose absorption in mouse intestine. Bioassay fractionation gave a fatty acid, 9,12,13-tryhydroxy-10(E)-15(Z)-octadecadienoic acid identified by spectral analysis. The Peach leaf may be a new resource for preventing metabolic disorders such as diabetes. Peach leaf will be expected to apply as the health food and to contribute to maintenance for people's health in the near future.

抄録全体を表示

Systematic phytochemical screenings have been performed on land plants with attention paid to their glycoside constituents, which have resulted in the isolation of a variety of steroidal glycosides from Liliaceae plants and triterpene glycosides from Ranunculaceae plants. In our continuous studies on new highly-polar steroidal and triterpene glycosides from Liliaceae and Ranunculaceae plants, we have now examined the fresh bulbs of Allium karatavience and the fresh tubers of Eranthis cilicica. Five steroidal bisdesmosides (1-5), including four new compounds (1-4), were obtained from the MeOH extract of the bulbs of A. Karatavience, and two new triterpene bisdesmosides (6,7) were isolated from that of the tubers of E. cilicica. The structure of the isolated compounds were elucidated on the basis of extensive spectroscopic analysis, including that of the high-resolution NMR (^1H-^1H COSY, PHNOESY, HMQC, HMBC, 1D-selective-TOCSY, and HSQC-TCOSY) data, and the results of acidic or enzymatic hydrolysis. To the best our knowledge, 1 is the first representative furostanol glycoside, having a β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl moiety at the C-26 position of the aglycon. The linear hexaglycoside attached at C-28 of the aglycon in 6 and the branched tetraglycoside attached at C-3 of the aglycon in 7 have not been reported as sugars of either triterpene glycosides or steroidal glycosides. It is also notable that 7 has been found to contain D-allopyranose as a sugar component, which is rarely encountered in the plant glycosides.

抄録全体を表示

Cremanthodium plants grow in the high mountain area of northwest and southwest region of China. They are used as Tibetan traditional herbs. Due to the difficulty of the plant collection only a few reports have been published so far. Chemical constituents, as well as their base sequences in atpB-rbcL intergenic region, of Cremanthodium helianthus, C. angustifolium, C. lineare var. lineare, and C. campanulatum, are investigated. Thirteen secondary metabolites were isolated and their structures were established by spectroscopic methods, especially 2D NMR spectra. All of seven compounds (1-7) isolated from C. helianthus, C. angustifolium, and C. lineare var. lineare were known eremophilane-type sesquiterpenoids, which were quite often discovered in Ligularia plant, closely related to genus Cremanthodium and widely distributed around the Hengduan Mountains. On the other hand, C. campanulatum contained four bisabolane-type sesquiterpenoids (8-11), one oplopane derivative (12), and one lignan compound (13), four of which were new. C. campanulatum seems to be related to L. lankongensis because it also produces the bisabolane-type compounds but not eremophilanes. In the base sequence analysis of five Cremanthodium samples, different genetic types were observed even in two C. angustifolium samples. Although the examples are few, intra-specific diversity, along with inter-specific diversity may be present in Cremanthodium species.

抄録全体を表示

Strychnos cocculoides is a widely distributed plant in tropical regions. Its root barks are locally used in Tanzania folk medicine for the treatment of snake bites, fever and stomach pain. Strychnos species are reputed to their wide distribution of compounds such as trimeric indolomonoterpenic alkaloids (Phillippe et al., 2003). The present studies were carried out on methanolic extracts from stem and roots of Strychnos cocculoides. We have isolated the new bis-iridoid glucoside, composed of two known compounds 3 and 4, and named Cocculoside (1). We also isolated some known compounds, a phenolic glucoside, 2-Hydroxy-3-O-β-D-glucopyranosyl-benzoic acid (2) (Rashid et al., 1996), together with iridoid glucosides, Loganic acid (5) (Zhang et al., 2003), Morroniside (6) (Otsuka et al., 2001), and a iridoid monoterpene, Sarracenin (7) (Miles et al., 1976). Compound 6 was isolated as a major component and observed in the ^1H NMR spectrum as a mixture of the anomeric forms. Morronoside (6) and Sarracenin (7) have been isolated from plant of the genus Strychnos for the first time.

抄録全体を表示

We have been studying on the ethnological herbal medicines used by ethnic minority groups in Yunnan Province, China, aimed at searching new natural products leads for therapeutic agents. As a part of this study, we have investigated on the roots of C. spicatus collected in Yunnan Province, which have been used by the Dai ethnic group for the treatment of high-blood pressure in combination with several herbal plants. Two new and a known compounds were isolated from the EtOAc-soluble fraction from the roots of C. spicatus. The structures for these compounds were characterized by the 1D- and 2D-NMR examinations and their absolute sterostructures were elucidated by CD spectroscopic analysis. These compounds are dimeric lindenane-type sesquiterpenes, in which two lindenane units are bonded at C-6-C-8' and C-15-C-9' by endo Dields-Alder cycloaddition, with a unique 18-membered macrocyclic trilactone ring. We have also studied on the C. spicatus roots, cultivated in Japan, which has resulted in the isolation of four new dimeric lindenane-sesquiterpene with an 18-membered macrocyclic trilactone ring, a new dimeric lindenane-sesquiterpene with an acetyl group, and a new indenane sesquiterne, together with four known dimeric lindenane-sesquiterpenes. The structures for these new compounds were also elucidate by detailed spectroscopic examinations. Among the isolated compounds, two compounds, isolated from the roots of C. spicatus cultivated in Japan, contained a perhydroxide group in the molecular. They are the first examples of dimeric lindenane-sesquiterpenes possessing a perhydroxide group.

抄録全体を表示

Based on the fact that some of the bioactive materials isolated marine animals have been produced by bacteria, we have focused our attention on new antitumour materials from microorganisms inhabiting the marine environment. As part of our ongoing seach for new antitumour metabolites produced by microorganisms from marine organisms, chaetomugilin A (1)-H (8) have been isolated from a strain of Chaetomium globosum 106B-6 originally separated from marine fish Mugil cephalus. The absolute stereostructures of 1-8 have been elucidated on the basis of spectroscopic analyses using 1D and 2D NMR techniques, some chemical transformations and X-ray crystal structure analysis. Compounds 1-8 were examined together with 5-FU as standard sample in the cytotoxic assay using the murine P388 leukemia cell line and the human HL-60 leukemia cell line. Compounds 3 and 6 exhibited significant cytotoxic activity against P388 and HL-60 cell lines equal to 5-FU. In addition, 1 and 3 were examined using a disease-oriented panel of 39 human cell lines. The effective concentration (MG-MID) of both compounds were a little higher, but the delta and range values of 1 (1.13 and 1.24, respectively) and 3 (0.71 and 1.19, respectively) disclosed that these compounds showed selective cytotoxic activity (effective value: delta&ge;0.5 as well as range&ge;1.0).

抄録全体を表示

The assignment of ^1H and ^<13>C NMR signals of a complex type triantennary asialooligosaccharide was examined using 2D selective-TOCSY-DQFCOSY and HSQC-TOCSY experiments. The 2D selective-TOCSY-DQFCOSY experiment exhibits a 2D DQFCOSY spectrum of an individual monosaccharide in the undecasaccharide, although the NMR signals of several monosaccharides in the triantennary undecasaccharide are heavily overlapped. Selective excitation of each anomeric proton signal and subsequent TOCSY experiment afforded transverse magnetization corresponding to all of the proton signals of the monosaccharide. This magnetization was then developed with the corresponding DQFCOSY pulse sequence to afford the DQFCOSY spectrum of the individual monosugars. In order to assign ^<13>C signals, a conventional 2D HSQC-TOCSY spectrum was examined and compared with an unambiguous assignment of 2D selective-TOCSY-DQFCOSY thus obtained. This systematic analysis made it possible to obtain an assignment of the ^1H and ^<13>C NMR signals of the triantennary undecasaccharide. In addition, these experiments also revealed all of the glycosyl positions in the triantennary undecasaccharide.

抄録全体を表示

Marine organisms produce various molecules with remarkable physiological activities. As part of our continuing chemical studies of marine organisms, we examined the constituents of the cyanobacteria collected at Okinawa prefecture, whose crude organic extracts shows cytotoxicity against HeLa S_3 cells. Bioassay-guided fractionation of the extracts led to the isolation of novel macrolide glycoside 1. The molecular formula of macrolide glycoside 1 was determined to be C_<34>H_<52>O_9 on the basis of HR-ESIMS and NMR data. The gross structure of 1 was established on the basis of spectroscopic data. The geometry of olefins were determined based on ^<13>C chemical shifts of adjacent methylenes or methyls to the olefins. Relative stereochemistry of sugar moiety was determined by analysis of vicinal ^1H-^1H coupling constants. Macrolide glycoside 1 was an 18-membered macrolide and showed cytotoxicity against HeLa S_3 cells, with an IC_<50> value of 1μg/mL. To determine the stereochemistry of 1, degradation reaction of 1 such as ozonolysis have been carried out.

抄録全体を表示

Bromopyrrole alkaloids are known to be one of the most common metabolites contained in marine sponges. During our search for bioactive substances from marine organisms, we have isolated bromopyrrole alkaloids with unique cyclic skeletons from sponges of the genus Agelas or Hymeniacidon. Recently, three new bromopyrrole alkaloids, nagelamides K, M, and N (1-3), have been isolated from an Okinawan marine sponge Agelas sp. (SS-1134). The structures and relative stereochemistry of 1〜3 were elucidated on the basis of spectroscopic data. Nagelamide K (1) is a new dimeric bromopyrrole alkaloid possessing a rare piperidinoiminoimidazolone ring with an aminoimidazole ring and a taurine unit. Nagelamide M (2) is a novel bromopyrrole alkaloid possessing an 2-amino-octahydropyrrolo[2,3-d]imidazol ring with a taurine unit, while nagelamide N (3) is a new bromopyrrole alkaloid consisting of an 2-amino-tetrahydroimidazol-4-one ring with a taurine unit and 3-(dibromopyrrole-2-carboxamido)propanoic acid moiety. Nagelamides K, M, and N (1-3) showed modest antimicrobial activity.

抄録全体を表示

Constituents of the glandular trichome exudates on the leaves of Paulownia tomentosa (Scrophulariaceae) and the leaves and stems of Ibicella lutea and Proboscidea louisiana (Martyniaceae) have been investigated. P. tomentosa exudates afforded 30 acylglycerols which belonged to the 1,3-diacetyl-2-fattyacylglycerol, 1-acetyl-2-fattyacyl-sn-glycerol, and 2-fattyacylglycerols families, wherein the fattyacyl moiety was either eicosanoyl or octadecanoyl group bearing hydroxy and/or acetoxy groups at the 3-, 3,6-, 3,7-, 3,8- or 3,9-positions. The configurations of the stereogenic centers in the fattyacyl moieties were determined by ^1H NMR analysis of the mono-(R)- and (S)-2-naphtyl-2-methoxyacetate derivatives of the fatty acid methyl esters. The configuration at C-2 of the glycerol moiety of the 1-acetyl-2-fattyacylglycerols was determined to be S by chemical conversion to a stereodefined mono-TBDPS-glycerol. Relatively abundant constituents included 1-acetyl-2-(3R-acetoxy-6S-hydroxyeicosanoyl)-sn-glycerol, 2-(3R,8R-diacetoxyeicosanoyl)glycerol, 2-(3R,9R-diacetoxyeicosanoyl)glycerol, 2-(3R-acetoxy-6S-hydroxyeicosanoyl)glycerol, 2-(3R-acetoxy-8R-hydroxyeicosanoyl)glycerol and 2-(3R-acetoxy-9R-hydroxyeicosanoyl)glycerol. I. lutea exudates yielded glycolipids, 6S-(6-O-acetyl-β-D-glucopyranosyloxy)eicosanoic acid and 6S-(β-D-glucopyranosyloxy)eicosanoic acid and their C_<18> and C_<22> acid analogs, 2-acylglycerols, 2-(3R-acetoxynonadecanoyl)glycerol and 2-(3R,6S-diacetoxynonadecanoyl)glycerol and their C_<17>, C_<18>, C_<20> and C_<21> acid analogs, along with their isomers having iso- and anteiso-fattyacyl structures. In addition, a new dammarane triterpene, betulatriterpene C 3-acetate, was isolated. P. louisiana exudates furnished glycolipids, 8S-(6-O-acetyl-β-D-glucopyranosyl-oxy)eicosanoic acid, 8S-(β-D-glucopyranosyloxy)eicosanoic acid, their C_<22> acid analogs, and 6S-(β-D-glucopyranosyloxy)eicosanoic acid, 2-acylglycerols identical to those found in I. lutea, and 11 new dammarane triterpenes, such as (1β,3α,12β,20S)-dammar-24-ene-1,3,12,20-tetrol and (3α,11α,12β,20S,24R)-24-hydroperoxydammar-25-ene-3,11,12,20-tetrol.

抄録全体を表示

Cellular slime molds are thought to be excellent model organisms for the study of cell and developmental biology because of their simple pattern of development. However, there have been no reports on secondary metabolites of them. We have focused on the utility of cellular slime molds as novel resources for natural product chemistry, and have studied the diversity of secondary metabolites produced by them as well as their physiological and pharmacological activities. In this study, we show the isolation of five novel secondary metabolites Pt-1〜5 (1-5), which are prenylated or geranylated aromatic compounds, from cellular slime mold Polysphondylium tenuissimum. The structures of 1-5 were determined by spectral means including EIMS, and ^1H and ^<13>C NMR. In addition, we synthesized DIF-1 (6), a stalk-cell differentiation inducer in cellular slime mold Dictyostelium discoideum, and its analogs. DIF-1 (6) also exhibited anti-proliferative activities and induced cell differentiation in mammalian cells. We evaluated their biological activities to assess structure-activity relationship of them and to develop useful compounds for the study of both Dictyostelium development and cancer biology.

抄録全体を表示

There is substantial interest in discovering new lead molecules from natural products for agricultural application including plant growth regulators. In this study, the marine-derived bacterium Bacillus subtilis was found to produce two new isocoumarins, bacilosarcins A (1) and B (2) as well as three previously reported isocoumarins, amicoumacins A, B, and C. Isocoumarin-type metabolites from microorganisms are characterized by the amino-containing substituent at 3-position in the dihydrocoumarin core. There have been several such isocomarin compounds as baciphelacin and amicoumacins isolated from bacteria of the genus Bacillus. The structures of 1 and 2 were assigned on the basis of NMR analysis and chemical conversions. 1 possesses an unprecedented 3-oxa-6,9-diazabicyclo[3.3.1]nonane ring system whereas 2 has a 2-hydroxymorpholine substructure that is rare in nature. The new compounds showed plant growth inhibitory activity against barnyard millet. We herein describe the isolation, structure elucidation, absolute stereochemical assignment, and biological activity of 1 and 2.

抄録全体を表示

Influenza virus infection is widespread and often critical for patients with respiratory diseases, immunosuppressive syndromes, aged persons with cardiopulmonary diseases and so on. Recent human infections caused by highly pathogenic avian influenza virus posed a pandemic threat. During screening for anti-influenza virus compounds from microbial origins, a novel antibiotic, wickerols, produced by a fungus, Trichoderma atroviride FKI-3849, isolated from a soil sample from Hawaii Island, Hawaii, USA, exhibited anti-viral properties. The strain was cultured in Erlenmeyer flasks on a rotary shaker at 28℃ for 4 days. Six liters of the cultured broth was extracted by ethyl and then extracted by ethyl acetate. This was chromatographed over a silica gel column, and 18.1mg of wickerol A and 3.2mg of wickerol B were obtained as white powders. The structures of wickerol A and B were elucidated by NMR study. They have a unique 3H-5a,9-methanocycloocta[cd]indene skeleton (fused 6-5-6-6 ring system). The structures were confirmed by X-ray crystallographic analysis. Wickerol A inhibited replication of mouse-adapted influenza virus A/PR/8/34 in Madin-Darby canine kidney (MDCK) cells at an IC_<50> value of 0.07μg/ml, while it inhibited the growth of MDCK cells at an IC_<50> value of 7μg/ml. Wickerol B showed week anti-influenza acitivity compared with wickerol A.

抄録全体を表示

Chlorosulfolipids (CSLs) are unusual naturally occurring compounds, which were first isolated at the end of the 1960s from freshwater microalgae such as Chrysophyceae Ochromonas danica and Poterioochromonas malhamensis. They constitute 15% of total lipids. The preparation of the CSLs in sufficient purity for structural characterization has proven to be difficult because the polar lipids were extremely hydroscopic and viscously. Therefore, any bioassays have not taken in nature form and structural elucidation was not satisfied with stereochemical analysis. Herein, we report successful isolation and determination of relative and absolute configuration of eight chlorosulfolipids. The Chrysophyta O. danica (IAM CS-2) was cultured at 25℃ for 1 week in a medium containing 0.1% glucose, 0.1% tryptone, 0.1% yeast extract and 0.05% meat extract with aeration. The cells were collected by continuous centrifugation, and freeze dried. The freeze-dried cells were homogenized and extracted with 1L of MeOH (three times) and 1L of EtOAc. The combined extracts were evaporated in vacuo and sequentially extracted with hexane, CHCl_3, EtOAc, BuOH and water. The active fractions (EtOAc, BuOH) of brineshrimp assay were applied repeatedly to silica gel and flash ODS column chromatography. Finally reverse phase-HPLC using a C_<30> column and an ELSD detector yielded four known CSLs (1,6-8) and four new compounds (2-5). Compound 1 was a major component of CSL from O. danica. Planar structure of 1 was established by HR-ESI-MS, 1D, 2D NMR and ESI-MS/MS. By using the JBCA method, relative configurations of 1 were determined by measuring ^<23>_J_<C,H> from HETLOC and J-IMPEACH-MBC spectrum. The absolute configuration was determined by modified Mosher's method after the sulfatide was hydrolyzed. In conclusion, 1 was identified as 2,2,11,13,15,16-hexachlorodocosane 1,14-disulfate. In the same manner, structures of 2-8 (2: 2,2,11,13,15,16-hexachloro-14-docosanol 1-sulfate, 3: 2,2,11,13,15,16-hexachlorodocosane 1,14-diol, 4: 11,13,15,16-tetrachlorodocosane 1,14-disulfate, 5: 11,13,15-tri-chlorodocosane 1,14-disulfate, 6: 13-chlorodocosane 1,14-disulfate, 7: docosane 1,14-disulfate, 8: 14-chlorotetracosane 1,15-disulfate) were determined.

抄録全体を表示

Marine dinoflagellates are known to produce bioactive secondary metabolites. Members of Amphidinium are among the most abundant and diverse sand-dwelling benthic dinoflagellates world-wide, and have been proven to be important sources of structurally unique polyketides. Recently, we have isolated three new 20-membered macrolides, iriomoteolides-1a〜1c, from a marine benthic Amphidinium sp. (strain HYA024). Further examination of the extract led to the isolation of two new odd-numbered macrolides, iriomoteolides-2a (1) and 3a (2). The cultured algal cells of the Amphidinium HYA024 strain were extracted with McOH/toluene. The toluene-soluble fractions of the extract were subjected to SiO_2 gel, C_<18>, and then NH_2-SiO_2 columns. Macrolide-containing fractions were separated by C_<18> HPLC to afford two new odd-numbered macrolides, iriomoteolides-2a (1) and 3a (2), together with iriomoteolides-1a〜1c. Structure elucidations of 1 and 2 were carried out using detailed analyses of 2D NMR spectra. The relative and absolute stereochemistries for 1 were assigned by combination of conformational analyses using NMR data and modified Mosher's method of 1 and its reductive product. On the other hand, those for 2 were elucidated on the basis of conformation studies of 2 and its acetonide and modified Mosher's method. Iriomoteolide-2a (1) is the first natural product possessing a 23-membered macrolactone ring consisting of a successive polyketide chain, while iriomoteolide-3a (2) is a new 15-membered macrolide having an allyl epoxide, three hydroxyl groups, and two methyl branches. Iriomoteolides-2a (1) and 3a (2) exhibited cytotoxicity against human B lymphocyte DG-75 cells (IC_<50>:0.006, and 0.08μg/mL, respectively).

抄録全体を表示

We reported the structures of four prenylated indole alkaloids, notoamides A-D (1-4), which isolated from a marine-derived fungus Aspergillus sp. Although the starting substrate for 1-4 was deduced to be notoamide E (7), it was not found in the metabolites of the fungus. Then, we investigated the presence of 7 in the culture and found that it existed only in the culture incubated for five days and disappeared the next day. These results clearly indicated that 7 was produced by the fungus in the early stage and immediately converted to other metabolites. Subsequently, we carried out the feeding experiments with doubly ^<13>C-labelled 7 and obtained 3, 4, and 8-11. However, contrary to our expectation, compounds containing a bicyclo [2.2.2]diazaoctane ring such as 1 and 2 were not obtained at all. Thus, the biosynthesis of the indole alkaloids containing the bicyclo [2.2.2]diazaoctane ring is an interesting enigma, and the search for the progenitor of 1 and 2 is under investigation. Additionally, we isolated ten new alkaloids 12-21, which showed the structural diversity, and found that 21 was an opposite enantiomer of versicolamide B, which was isolated from the closely related fungus by Glores et al. They already reported the isolation of opposite enantiomers of 2 and 5. It is interesting to know the biochemical pathways involved in the selective production of three opposite enantiomers by these two fungi.

抄録全体を表示

Snakebite accidents by the South American rattlesnake Crotalus durissus terrificus account for 10% of those occurred in Brazil. It induces severe neurological symptoms, but does not induce pain or severe tissue destruction at the site of inoculation, which is in contrast to the most other snakebites. Due to these properties, the crude venom of this snake used to be employed for controlling pain, for example, of cancer. Recent studies using the crude venom experimentally demonstrated that this venom shows antinociceptive effect more potent than morphine. This effect is orally active and long-lasting for 3-5 days, and despite mediated by opioid receptors, it dose not develop peripheral tolerance nor induce physical dependence unlike morphine. These remarkable properties prompted us to purify and chemically characterize the substance responsible for the analgesic effect. Bioassay-guided fractionation led to the isolation of a novel peptide, designated crotalphine, with a sequence of 14 amino acid residues having a single disulfide bond. We report herein the isolation, sequence determination and synthesis of crotalphine. Pharmacological evaluation using synthetic peptide will also be reported.

抄録全体を表示

Accidental ingestion of a toadstool, Russula subnigricans causes lethal poisoning to human. In the 1950's, the first poisoning caused by this mushroom was reported. Since then there have been no reports about lethal poisoning for 50 years, which was enough to raise doubts about its existence. However, in these three years, 2005 to 2007, the poisoning accidents were continuously happened and four people died. Although chemical studies on this fungus were reported using mushrooms distributed in Miyagi prefecture, the isolated compounds, russuphelins, russupherol, and hydroxybaikiain, have no toxicity on mouse. Accordingly, we studied the isolation of the toxic constituent of R. subnigricans. One of the reasons that such a strong toxin has not been revealed until now is the incomplete classification of this mushroom, that is, there are many resemble species distributed in Japan. We collected three species in Kyoto, Miyagi, and Saitama prefectures. The aforementioned compounds were found only in the Miyagi species. All three species show toxicity on mouse by intraperitoneal injection of the water extract; however, only the Kyoto species exhibits toxicity by oral injection. Accordingly, we estimated that the Kyoto species is the genuine R. subnigricans. During the separation steps, we found that the toxicity was remarkably decreased after concentration to dryness; therefore, all manipulations were carefully performed. The water extract was successively separated through ODS column chromatography, ion exchange chromatography, and gel filtration to give an aqueous solution of the toxic compound. The toxic compound was revealed to be unstable under concentration to dryness (polymerization occurs) and volatile, which was turned out to be the cause of decrease in toxicity after evaporation. The unstable toxin was converted to a stable derivative using diphenyldiazomethane. Taking ^1H, ^<13>C NMR and MS spectral analyses of the toxic compound and its derivative into consideration, the structure of the toxic compound was determined to be cycloprop-2-ene carboxylic acid. This compound was found only in the Kyoto species.

抄録全体を表示

Marine microorganisms are recognized as important sources of pharmacologically active metabolites, and a growing number of marine fungi have been reported to produce novel bioactive secondary metabolites. We report chemical investigation of the metabolites obtained from the extracts of two sorts of marine fungi culture filtrates. We previously reported three antibacterial chlorine-containing components of Aspergillus ostianus strain 01F313 that had been isolated from an unidentified marine sponge collected at Pohnpei. The chlorine must have originated from cultivation medium composed of natural seawater. Expecting that bromine-containing compounds might be obtained by the use of a medium, in which a bromide solution replaces seawater, we cultivated the same strain in a brominemodified 1/2PD medium. Actually, we were able to isolate the brominated compounds, and found, at the same time, that the metabolites were considerably different from those obtained from the strain cultured in seawater medium. We also succeeded in isolating several benzodiazepine alkaloids that are not produced in seawater medium. The structures of compounds 1 and 2 were revised from the previously-reported betaine structures to unique oxepin ones by X-ray crystallography. Concomitant known alkaloids, compounds 3, 4, and 5, and a new compound, compound 6, have a common circumdatin framework. Penicillium sp. is known as sources of hybrid polyketides such as berkeleytrione and berkeleydione. We detected cytotoxic activity against A549 cells (IC_<50> 500μg/mL) in an acetone extract of the marine-derived fungus, Penicillium minioluteum 03HE3-1, which had been isolated from sea mud of Heita Bay, Kamaishi, Japan. The fungus was cultured in a 1/2PD medium containing 50% seawater. Four novel hybrid polyketide-terpenoid metabolites were obtained from the culture filtrate. Their structures were determined by NMR spectroscopic analyses and X-ray crystallography. The biosynthetic pathway of four meroterpenes from farnesyl pyrophosphate and 3,5-dimethylorsellinic acid is proposed.

抄録全体を表示

In this symposium we will summarize our 6-year activity on construction of marine microbe library and present part of our results on the exploration of new bioactive molecules from our collection. The 6-year project entitled "Construction of a Genetic Resource Library of Unidentified Microorganisms", initiated by New Energy Industrial Technology Development Organization (Ministry of International Trade and Industry jurisdiction), aimed at establishing domestic sound research foundation to promote better utilization of microbial function in industry. As one of the contractors for this project, Marine Biotechnology Institute Co., Ltd. (Kamaishi, Japan) has focused on collecting microbes from marine environments. During 2002-2008, we have conducted more than 25 samplings along the coast of Japan Islands and around Micronesian Islands such as Yap, Palau, and Pohnpei, and constructed a comprehensive library of more than 25 thousands marine-derived microbes, with nearly one fifth being new at species level based on the 16S rDNA sequence. The library is mainly composed of proteobacteria belonging to Alpha- and Gamma- subclasses and Bacteroidetes group bacteria, but importantly includes many species from Phyla Planktomycetes, Verrucomicrobia, Chloroflexi, and Gemmatimonadates, which in the past the isolation have only been sparsely reported. Additionally, more than 500 of new fungi, based on the 28S rDNA sequence, were identified in our collection. The collected microorganisms were systematically cultivated to obtain their fermentation extracts and stocked in a 96-deep-well format to distribute as unique biomedical screening sources to our 20 collaborators from universities and companies. This extract library is also screened with our in-house assay settings to evaluate its biomedical potential. One of the promising strains that we encountered during the in-house antibacterial screening is HC35, which belongs to a recently proposed genus Rapidithrix within the Phylum Bacteroidetes. The strain was isolated with an E.coli-streaked seawater-agar plate method from subsurface silts of the muddy land alongside the Ariake Inland Sea, southwest Japan. Fermentation of strain HC35 in modified Marine Broth 2216 and bioassay-guided fractionation resulted in the isolation of novel antibiotics, ariakemicins A and B. The ariakemicins, a pair of positional isomers with regard to a double bond, are unusual linear polyketide-peptides marked by a oxazole ring, two highly unsaturated polyketide chains, and a terminal isovanilloylbutyl group, the latter two of which are connected through two enamide linkages. Antimicrobial testing against 7 strains composed of Gram-positive and Gram-negative bacteria and a yeast Candida albicans revealed that this chromatographically inseparable mixture was effective at killing Gram-positive bacteria, especially Staphylococcus aureus with the MIC of 0.46μg/mL. The discovery of ariakemicins represents the validity of focusing on untapped microbial sources for the exploration of bioactive molecules of novel structure classes.

抄録全体を表示

Sex pheromones have been identified from about 600 lepidopteran species, but information of chemicals secreted by species in the Arctiidae, one of the evolved families including various species. Particularly for the species in the Lithosiinae, a subfamily of the including, pheromones have never been reported. In order to understand structural diversity of the pheromones, we caught adults of several Lithosiinae species, using a black-light trap in the Iriomote Islands, located in a subtropical zone. Among the collected insects, females of Lyclene dharma dharma produced a 2:1:1 mixture of novel pheromone components (I-III), which strongly stimulated male antennae on a GC-EAD analysis. Using GC-MS analysis and chemical derivatizations, such as removal of a carbonyl function by the Wolff-Kishner reaction, the following structures were estimated: 6-methyl-2-octadecanone (I), 14-methyl-2-octadecanone (II), and 6,14-dimethyl-2-octadecanone (III). These methyl-branched ketones have not been identified as a natural product, indicating that they constitute a new chemical group of lepidopteran female sex pheromones. While the stereochemistry of the chiral centers could not be determined because it was difficult to collect a sufficient amount of a pheromone extract, their plain structures were confirmed by synthesis of racemic I-III starting from diols. The mass spectrum of each synthetic ketone coincided well with that of the natural component, and the 2:1:1 mixture of the racemates successfully attracted many males in a field in the Iriomote Islands. Furthermore, we are now examining to improve the synthetic yield and established another route via Wacker oxidation terminal alkenes, where all of the three components were constructed with common synthetic units.

抄録全体を表示

In the course of screening for endogenous flowering inducer, α-ketol of octadecadienoic acid (KODA, Figure1) was isolated from Lenma paucicostata. KODA showed the activity after reacting with norepinephrine (NE) under the basic conditions. Further investigations have revealed that FN1 is a major active compound, which is expected to be formed by cycloaddition between 12-olefin of KODA and α, β-unsaturated carbonyl of noradrenochrome, an oxygenated form of NE. However, the mechanism of flower-induction of FN has not been examined. In present study, we carried out the structure-activity relationship study of the fatty acid moiety of FN for flowering in L. paucicostata by using series of KODA analogs shown in Figure 2. Firstly, we synthesized nine fatty acid analogs, where single or combinative alterations have been made to the structural components of KODA and tested for their ability to induce flowering. With the exception of compounds 8, 10, 12, all these analogs proved to have a flowering activity after reacting with NE. Compound 4, in which 15-olefinic bond is saturated, displayed high activity of same magnitude as KODA. This suggested that olefinic bond at 15-position in KODA in not important for activity. Compounds 7 and 9 displayed a significant activity but less in magnitude as the parent KODA. This implied that 9-hydroxy group may not be involved in primary recognition of the target whereas the presence is favorable to show high activity. Compounds 5 and 6, in which their alkyl chains are shortened, displayed flowering activity at a concentration of more than 1μM. In addition to this, the biological result obtained with methyl ester 11 indicated that recognition of the aliphatic chain and terminal carboxy group in FNs is relatively obscure. On the other hand, compounds 8, 10, and, 12, changing of β, γ-unsaturated carbonyl moiety led to complete loss of activity in L. paucicostata. This result showed that cycloaddition of fatty acid with NE is inevitable process to induce flowering in L. paucicostata. Furthermore, to obtain clearly insight in the structural requirements of FN1 for flower inducing activity, we prepared FN derivatives from corresponding fatty acid analogs and evaluated their activity.

抄録全体を表示

We have been dealing with a hydrophobic vitamin B_<12>, heptapropyl cobyrinate perchlorate [Cob(II)7C_3ester] ClO_4, which has ester groups in place of the peripheral amide moieties of the naturally occurring vitamin B_<12>. In order to construct a good catalytic system, we prepared various artificial enzymes with vitamin B_<12> activities. One example is a vesicle-type artificial enzyme composed of peptide lipids and the hydrophobic vitamin B_<12>. Another one is a silica gel having vitamin B_<12> derivatives by the sol-gel method. In this work, we reported a new catalyst; human serum albumin (HSA) containing vitamin B_<12> derivatives. HSA is the most prominent protein in plasma, and serves as a transporter for hydrophobic molecules in vivo. We use HSA as an apoenzyme model for construction of an artificial B_<12> enzyme. The incorporation of hydrophobic vitamin B_<12> derivatives into HSA is primarily controlled by the hydrophobicity of the peripheral ester groups. Microenvironmental property around the hydrophobic vitamin B_<12> in HSA was examined by fluorescence and fluorescence polarization measurements. The hydrophobic vitamin B_<12> itself in HSA is in a microenvironment equivalent in medium polarity to dichloromethane. We added [Ru(II)(bpy)_3], as a photosensitizer, to a B_<12>-HSA artificial enzyme, and then carried out the degradation of 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT), which is characterized by a pronounced insecticidal property and has been used worldwide for the last several decades despite its known hazardous effects on human health and wildlife, mediated by hydrophobic vitamin B_<12> under irradiation with visible light. DDT was decomposed to DDD and DDE, which are dechlorinated compounds. This result is very interesting from the viewpoint of green chemistry.

抄録全体を表示

There are many kinds of flavonoids which have interesting bioactivities in nature. Although many synthetic methods of the flavonoids have been developed, these methods are not always effective for highly substituted flavonoides. Herein we report more effective synthetic method for flavonoids through a β-diketone intermediate, which enabled us to synthesize six unnatural flavonoids (1a-c, 2a-c) and natural flavone C-glycosides, Chafuroside A (3) and B (4). The key intermediate 7 was easily synthesized by coupling of an acetophenone derivative 5 and N-acylbenzotriazole 6 under basic condition. Removal of TBDPS group with TBAF and subsequent cyclization under acidic condition gave a flavonoid 8. Finally, deprotection of benzyl groups furnished the flavone 1a in 5 steps. On the other hand, the flavonol 2a was also obtained by oxidation of 8 with DMDO and subsequent deprotection. (Scheme 1) Interestingly, flavones 1c and flavonol 2c exhibited potent sialyltransferase inhibitory activities. (Table 1) Chafuroside A (3) and B (4) are unique flavonoids isolated as minor constituent of oolong tea extract showed potent anti-inflammatory activity. Structurally, these flavones are consisting of unusual fused tetracyclic ring system composed of mannosyl moiety, A, and C rings of flavones framework. In order to synthesize 3 regioselectively, two hydroxyl groups of an aryl C-glycoside 9 were protected with TBDPS group and benzyl group, which afforded 13. The β-diketone 14 was synthesized by coupling between 13 and 6 followed by deprotection of TBDPS group. After cyclization of 14 under acidic condition, deprotection of Benzyl groups and intramolecular Mitsunobu reaction yielded 3. (Scheme 3) Thus, Chafuroside A (3) has been synthesized in 7 steps with 32% overall yield from 9. According to similar route, Chafuroside B (4) has been also synthesized in 5 steps and 13% yield from 9. (Scheme 4)

抄録全体を表示

Microbial and plant-derived bioactive metabolites are a treasury of organic compounds having various structures and biological activities. Therefore. we are screening cellular signal transduction inhibitors from bioactive metabolites. We also design and synthesize signal transduction inhibitors. NF-kappa B is the transcription factor that promotes transcription of inflammatory cytokines, cell adhesion molecules, and inhibitor of apoptosis proteins. We have designed dehydroxymethylepoxy quinomicin (DHMEQ) based on the structure of epoxyquinomicin isolated from Amycolatopsis. DHMEQ showed potent anti-inflammatory and anticancer activities in animal models without showing any toxicity. Recently, we found that DHMEQ directly binds to p65 to inhibit the NF-kappa B functions. In the course of our further screening of NF-kappa B inhibitors, we isolated 9-methystreptimidone from Streptomyces. 9-methystreptimidone increased the apoptotic sensitivity of adult T-cell leukemia cells. In the present research, we looked for novel anti-inflammatory agents having rather simple structures. We looked for the compounds that inhibit LPS-induced NO production in a macrophage cell line RAW264.7. As a result, we found that DTCM-glutarimide, a novel derivative of 9-methylstreptimidone, inhibited the LPS-induced NO production. Then, we looked into the mechanism of inhibition. It inhibited LPS-induced expression of iNOS and COX-2. However, it did not inhibit IL-6 expression unlike most NF-kappa B inhibitors. We then confirmed that it did not inhibit LPS-induced NF-kappa B activation. It also did not inhibit the MAPK activation. On the other hand, iNOS induction is known to be dependent on a transcription factor, AP-1 consisting of c-Jun and c-Fos. We found that DTCM-glutarimide inhibited nuclear translocations of c-Jun and c-Fos. Thus, DTCM-glutarimide is considered to be a novel anti-inflammatory agent having a simple structure and unique mechanism.

抄録全体を表示

SPR imaging technique has been used to observe multiple interactions between non-labeled biological molecules and surface-bound molecules. We have recently developed a "photoaffinity-linking" protocol to enable the introduction of structurally diverse small-molecules on solid surface in a functional-independent manner. Here we describe the improvement of the photoaffinity thiol linker of our SPR imaging platform, which enabled us to determine the binding site of p62/SQSTM1 to p38 mitogen-activated protein (MAP) kinase. SPR imaging experiments using a new photoaffinity linker 2 to immobilize the peptides derived from p62 on gold substrate indicated that the domain comprising amino acids 164-190 of p62 binds to p38 directly. These SPR analysis data and empirical biologic data revealed that the binding site of p62 to p38 is the domain corresponding to 173-182. We also describe a screening study of small-molecules which have inhibitory activity of tau aggregation. Starting with 2011 compounds, 202 compounds were identified as tau-binding compounds by chemical array screening. Among them, 53 compounds were detectable by our photoaffinity SPR platform using linker 3. Finally, it was revealed that 42 compounds have inhibitory activity for tau aggregation.

抄録全体を表示

Potassium isolespedezate (1) is a leaf-opening factor which is effective for the leaf of Cassia mimosoides. The target protein of 1 was reported to be a motor cell which is located in the joint of leaf and stem. And also, we found a binding protein of 1 (210kDa) from the membrane fraction of Cassia motor cell. Now, we found another binding protein of 1 which is located in the cytosol of motor cell and would be a genuine target protein of 1 (83kDa) which is involved in the control of nyctinastic leaf movement of Cassia plant. By this finding, the 210kDa-membrane protein is deduced to be a membrane transporter for 1.

抄録全体を表示

Oolong tea is a semi-fermented tea, which has been manufactured from leaves of Camellia sinensis (L) O. Kuntze, same as non-fermented Japanese or Chinese green teas. Major components of fresh tea leaves are caffeine and catechins such as epigallocatechin-3-O-gallete (EGCG). During the fermentation process, polyphenol oxidase (PPO) in tea leaves oxidizes the catechins into theasinensin and oolongtheanin. In addition, oolonghomobisflavans are known as condensation products of catechins by heat treatment. The aim of this study is to quantify these minor polymerized polyphenols by LC-MS/MS, and address their biological activities. Quantification of polyphenols by LC-MS/MS Theasinensin (TSN), oolongtheanin gallate (OTNG), and oolonghomobisflavan (OHBF) were synthesized from EGCG. Two new trimers and tetramers of oolonghomobisflavan were synthesized along the previous synthesis method of OHBF, and then their structures were determined by MS and NMR spectroscopy. Quantification of these compounds was performed by LC-MS/MS in the mode of multiple reaction monitoring (MRM). Compounds other than TSN-A were of small quantities in oolong tea leaves. Quantification of polymerized polyphenols was not performed with HPLC UV monitoring, but by LC-MS/MS method. Nine kinds of oolong teas in different fermentation levels were extracted and analyzed. The very small amount of trimers and tetramers of OHBF were included in "Black oolong tea", therefore, it was necessary to make concentrated solutions of these by column separation, and quantified the real amount by analyzing them. Biological activities of polymerized polyphenols Pancreatic lipase inhibitory activity of oolong tea polymerized polyphenols (OTPP) and some catechin dimmers have been already reported. In this study, new trimers and tetramers also exhibited strong inhibitory activity. Besides OTPP, the polymerized polyphenols, TSN, OTNG, and OHBF also have α-glucosidase inhibitory activity. These results suggest that oolong tea prevents the elevations of triglyceride in blood from meal fat, as well as inhibit the absorption of sugars derived from carbohydrates.

抄録全体を表示

Chloroplasts, which are thought to have originated from cyanobacteria, have their own genetic system which is similar to that of the bacteria from which they were derived. Recently, bacterial alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a key regulatory molecule controlling the stringent response, was identified in chloroplasts of plant cells. Similar to its function in bacteria, ppGpp inhibits chloroplast RNA polymerase, suggesting that ppGpp mediates gene expression through the stringent response in chloroplasts. However, a detailed mechanism of ppGpp action in chloroplasts remains elusive. We synthesized 6-thioguanosine 5'-diphosphate 3'-diphosphate (6-thioppGpp) as a photoaffinity probe of ppGpp, thus enabling the investigation of ppGpp binding to chloroplast RNA polymerase. We found that 6-thioppGpp, as well as ppGpp, inhibits chloroplast RNA synthesis in vitro in a dose-dependent manner. Crosslinking experiments with 6-thioppGpp and chloroplast RNA polymerase demonstrated that ppGpp binds the β' subunit (corresponding to the Escherichia coli β' subunit) of plastid-encoded plastid RNA polymerase composed of α, β, β', β", and σ subunits. Furthermore, ppGpp did not inhibit transcription in plastid nuclei prepared from tobacco BY-2 cells, suggesting that ppGpp does not inhibit nuclear-encoded plastid RNA polymerase. These results demonstrate that ppGpp functions as a stringent factor, governing gene expression in chloroplasts.

抄録全体を表示

(-)-Ternatin (1) is a highly N-methylated cyclic heptapeptide that was isolated from the mushroom Coriolus versicolor during our continuing search for potential anti-obesity agents from natural resources such as mushrooms. In our previous presentation (2006), we reported the isolation, structure elucidation and synthesis of 1, which potently inhibited fat accumulation against 3T3-L1 murine adipocytes. To clarify the detailed mode of action of (-)-ternatin (1) with regard to fat-accumulation inhibition in adipocytes, we started bioorganic studies of 1 and its analogues. We achieved the solution-phase synthesis of 1 and then evaluated its in vivo biological activity. Treatment with 1 at 5mg/kg/day was found to suppress the increase in body weight and fat accumulation in diet-induced obese mice. Due to the course, a structure-activity relationship (SAR) study of 1 was first investigated aimed at the recognition of the importance of side chain functionalities as well as a suitable site for advanced functionalization in its structure, e.g., biotinylation and introduction of a fluorescent unit.

抄録全体を表示

Accumulating data suggest that quorum-sensing system possesses a variety of roles in pathogenesis of infections. Recent progress of this field demonstrated that Pseudomonas aeruginosa autoinducers may have bactericidal activity to certain bacterium, although the understanding of the spectrum of activity and the mechanisms of action is limited. The present study examined potential of bactericidal activity of the autoinducers and their degradation product tetramic acids against Clostridium difficile, an anerobic gram-positive bacterium and a cause of antibiotic-associated pseudomembrane colitis. Methods: P aeruginosa autoinducers and a panel of tetramic acid derivatives were chemically synthesized. Clinical isolates of C. difficile were incubated in broth under anerobic condition in the presence of the above compounds. The structure-activity correlation and the mechanisms of action were examined in several ways, such as time-kill assay and observation of changes of morphology by electron microscopy. Results: Some tetramic acid induced more than 3 log reduction of viability of C. difficile within 30min at a concentration of 50μM. The cell surface was suggested to be one of the targets for bactericidal activity of tetramic acid, because the disturbance of cell membrane was demonstrated by electron microscopy after exposure to tetramic acid. It is likely that enol structure of tetramic acid may be essential for antibacterial activity of this molecule. Interestingly, the simultaneous presence of tetramic acid at sub-MIC dramatically reduced the MICs of aminoglycoside amikacin against C. difficile from >256μg/ml to 0.5μg/ml. Conclusions: These results demonstrate potential of P. aeruginosa autoinducer-derived tetramic acids as a novel antimicrobial agent against C. difficile.

抄録全体を表示

Some sea stars have the ability to detach the arm when they are attacked by a predator, wounded in the arm, or subjected to inferior circumstances (pH, air supply, etc.). Japanese Asterias amurensis is known to have this ability of autotomy. The body fluids of scalded A. amurensis induced an arm autotomy of the intact A. amurensis by intracoelomic injection into an arm. This response is divided into two types, "slow autotomy" and "quick autotomy", according to the time taking to detach the arm. Bioassay-guided separation of lyophilized body fluids by size-exclusion column chromatography and repeated HPLC (ODS) gave an active fraction, which induced the "slow autotomy". A mixture of nicotinamide (NA) and N-methylquinolinic acid (NMQA) were assigned in this fraction as the inducers of the autotomy. NMQA is suggested to be the substrate of NMDA glutamate receptor, which has not yet been detected in sea stars. The "slow autotomy" is, therefore, induced by the stimulation of excitatory nerve system through an NMDA glutamate receptor and a pathway implicated with NA. On the other hand, the "quick autotomy" is induced by the above two pathways when the stimulation of an AMPA glutamate receptor and the other unidentified pathways are simultaneously occurred. SALMFamide S1 is suggested to be the substrate of the AMPA glutamate receptor.

抄録全体を表示

Tuberculosis (TB) is one of the most common causes of morbidity and death in HIV-positive adults living less-developed countries. Eight million new TB cases and two million deaths by TB are estimated each year. It is now generally accepted that the requirement for minimum 6 months treatment for TB is due to the difficulty in eradicating non-replicating persistent Mycobacterium tuberculosis in the granuloma of infected region. Therefore, the lead compounds, which are effective to M. tuberculosis in both active state and dormant state, are urgently needed. It is also important to explore new targets for drug against M. tuberculosis in the dormant state. Then, we established a screening system in hypoxic condition inducing dormant state based on environment of infected region. Resulting the screening, halicyclamine A was re-discovered as a lead for anti-tuberculosis agent from a marine sponge of Haliclona sp. on the guidance of the constructed bioassay. Halicyclamine A showed growth inhibition against M. smegmatis and M. bovis BCG with MICs in the range of 1.0μg/ml-2.5μg/ml under both aerobic condition and hypoxic condition inducing dormant state, and the growth inhibitory activity of halicyclamine A was bactericidal. In order to elucidate action-mechanism of halicyclamine A, transformants of M. smegmatis, which over-expressed the genes of M. bovis BCG randomly, were prepared by gnomic DNA library of M. bovis BCG. As a result, the target molecule of halicylamine A was clarified to be included in the genome of M. bovis BCG from 2920.549kb to 2933.210kb.

抄録全体を表示

We have been studying mechanisms underlying a blue coloration of tulip, Tulipa gesneriana cv. Murasakizuisho that has blue color in the cells at the bottom of the inner perianth and purple color in the cells of the upper part. We purified the petal pigment and co-pigments and the structure was determined to be delphinidin 3-O-rutinoside (1), manghaslin (2), rutin (3), and mauritianin (4). From blue and purple part of the petals we prepared each colored protoplast and analyzed vacuolar pH, content of anthocyanin and co-pigments, and metal elements of them. The vacuolar pH values of the purple and blue protoplasts were 5.5 and 5.6, respectively; without any significant difference. The content of pigment and co-pigment was also the same. However, the Fe content in the blue protoplast was 25 times higher than that in the purple protoplasts. Furthermore, we could reproduce the blue and purple solution by mixing 1-4 with/without Fe^<3+>, respectively. We clarified that Fe^<3+> is essential for blue color development in the tulip (Shoji et al., PCP, 48, 2007). These results strongly suggested of vacuolar iron transporter in blue color development of tulip petals and now the study is in progress.

抄録全体を表示

Lipid components from 12 nonpathogenic Mycobacterium species were analysed. A novel cyclic C_<35>-terpene, named heptaprenylcycline 1, was obtained from 3 species, while octahydroheptaprenol 2, which has 3 Z-double bonds, was obtained from 6 species. The amounts of 1 and 2 in the cultured cells increased after the 4- to 6-d stationary phase. The yield of 1 was considerably greater at a higher temperature of 37℃ than at an optimal growth temperature of 28℃, while that of 2 remained unchanged at all the temperatures (20-40℃). A feeding experiment with D-[1-^<13>C] glucose revealed that 1 was produced via isopentenyl diphosphate, which is a metabolite of glycolysis and the methylerythritol phosphate pathway. The conversion of octahydroheptaprenyl diphosphate 2-PP to 1 was successful by using the cell-free extracts of M. chlorophenolicum, demonstrating that 2-PP is the biosynthetic intermediate of 1. This is the first example of the biosynthesis of a natural terpene via the cyclisation of a linear C_<35>-isoprenoid. The substrate 2-PP has Z-type prenyl moieties; however, terpene cyclases usually employ E-type isoprenoids. The gene encoding the terpene cyclase that cyclises prenyl diphosphate containing Z-double bonds has not been reported hitherto. Despite a careful search using the FASTA3 program, we could not detect any gene homologous to the known diphosphate-triggered type of mono-, sesqui- and diterpene cyclases in the genome of M. vanbaalenii, the DNA sequence of which has recently been elucidated. This suggests that a novel type of terpene cyclase might exist in the nonpathogenic Mycobacterium species.

抄録全体を表示

The red pigment Carthamin, the water-soluble yellow components (2), (3), (4) and (5) are the typical coloring matters included in safflower (Carthamus tinctorius L.). These coloring matters have a unique quinochalcone glucoside skeltone in their molecules and have been found only in safflower. In the previous papers, we elucidated that (3) was transformed to other coloring matters (2), (4), (5) and (6) in cultured safflower cells. In order to elucidate the biosynthetic pathway of these unique coloring matters in safflower, biosynthetic route of key compound (3) was investigated. At first, the synthesis of compound (7) and subsequent oxidation of (7) to the quinochalcone glucoside (monomer, 6) were investigated. However, very small amount of (6) was detected by air oxidation of (7). On the other hand, the reaction of (2) with (6) gave (3), and the reaction of (6) with glucose gave (2) in good yield respectively. These results indicate that the true key compound of this biosynthetic route is monomer (6). Hence, the synthetic route of (6) was then investigated. As a result of various experiments, it became apparent that the compound (11) was oxidized to (12) and the reaction of (12) with p-coumaric acid gave (6) in safflower cells.

抄録全体を表示

Terpenoids are widely distributed in nature. They include hormones that regulate life cycles of mammals, insects and plants. Preparation of labeled-compounds will be powerful tools for studying comprehensive analysis and biosynthesis of terpenoids. We synthesized fully C-13 labeled mevalonate with enzyme cocktails which contain recombinant four enzymes responsible for mevalonate biosynthesis from acetate, cofactors, and the substrate [U-^<13>C_2) acetate (99% C-13 label). The mass spectra of C-13 labeled mevalonolactone, as dehydration form of mevalonate, showed no dilution with natural abundance peaks. The NMR signals showing C-C coupling were observed from 0.7mg of the sample. To produce polycyclic terpenoids from acetate in one test tube, these four enzymes were combined with an enzyme cocktail for synthesis of prenyl diphosphate from mevalonate, and terpene cyclase. For example, amorphadiene is a biosynthetic intermediate of antimalarial reagent artemisinine. The amorphadiene is biosynthesized from farnesyl diphosphates by amorphadiene synthase, a sesquiterpene cyclase in Artemisia annua L. We cloned amorphadiene synthase open reading frame and produced its recombinant protein in E. coli. This cyclase protein was added into the enzyme cocktail responsible for labeled farnesyl diphosphates synthesis, and uniformly C-13 labeled amorphadiene was synthesized in one test tube. Similarly, addition of bifunctional ent-kaurene synthase protein into the enzyme cocktail synthesizing geranylgeranyl diphosphates led [U-^<13>C_<20>] ent-kaurene production. Thus, we established the basis of enzymatic synthesis of fully C-13 labeled multicyclic terpenoids in one test tube. These labeled probes will be used as functional analysis of unknown genes and comprehensive analysis of terpenoid biosynthetic pathways.

抄録全体を表示

Higher plants accumulate a wide range of glycosides as secondary metabolites, and are capable of conjugating sugar residues not only to endogenous metabolic intermediates but also to xenobiotics. The glycosylation reactions are catalyzed by plant UDP-glycosyltransferases, which transfer sugar moieties from UDP-activated sugar donors to low molecular weight acceptors. Glycosyl conjugation of lipophilic small molecules is an efficient tool to enhance water solubility, to improve stability and thereby to increase bioavailability and modify biological activity. Curcumin is a yellow pigment of turmeric (dried rhizome of Curcuma longa, Zingiberaceae). Although curcumin is an interesting compound as a novel medicine or a lead compound, its low water solubility limits further pharmacological exploration and practical application. Here we investigate that Catharanthus roseus cell suspension cultures are capable of converting exogenously supplied curcumin to a series of unnatural glucosides. The water solubility of curcumin glucosides is increased up to 20-million-fold compared with that of the aglycone, indicating that glucosylation of hydrophobic compounds is a powerful method to enhance water solubility. Furthermore, we describe the isolation of five cDNAs (Catharanthus roseus UDP-glucose glucosyltransferase CaUGT1〜5) from a cDNA library of cultured C. roseus cells, using a homology-based PCR cloning strategy directed at the conserved domain, the Plant Secondary Product Glycosyltransferase (PSPG) motif. Functional expression of the gene products in Escherichia coli demonstrated that CaUGT2 converts curcumin to curcumin monoglucoside and diglucoside, whereas CaUGT3 converts curcumin monoglucoside to curcumin monogentiobioside, gentiotrioside, and gentiotetroside.

抄録全体を表示

Actinorhodin (ACT) produced by Streptomyces coelicolor A3(2) is a member of benzoisochromanequinone (BIQ) antibiotics. ActVA-6 has been suggested to be involved in oxygenation at C-6 position of BIQs. However, actVA-6 homologous genes could not been found in the other BIQ clusters and instead actVA-5-type oxygenase genes are commonly present. This study deals with the possible contribution of actVA-5, actVA-6 and related actVB genes in ACT biosynthesis. A deletion mutant of the actVA-6 gene (ΔactVA-6) produced almost comparable amount of ACT compared with wild type strain, whereas ΔactVA-5,6 abolished ACT production and yielded a novel dimeric shunt product, actinoperylone (ACPL), with two chiral centers at C-3 and C-15 of each monomeric unit. Production of ACPL was also demonstrated by the introduction of the minimal gene sets for 6-deoxy-dihydrokalufungin (DDHK) in S. coelicolor act-deficient mutant. All these results demonstrate the critical role of ActVA-5 in C-6 oxygenation and derivation of its substrate DDHK from (S)-DNPA by ActVI-2 catalyzed stereospecific enoylreduction at C-15. Although Valton et al recently proposed that ActVA-5/ActVB is a two-component flavin-dependent monooxygenase system introducing the oxygen at C-8 of dihydrokalufungin (DHK), an essentially similar assay conditions converted emodinanthrone, a model substrate for C-6 oxygenation, into emodin. The same activity was detected with ActVA-6 but with less efficiency. Deletion of actVB resulted in the simultaneous production of ACT and ACPL, indicating that the C-6 oxygenation in vivo is indeed affected by the disorder in ActVA-5/ActVB system. In summary, the present study concludes that C-6 oxygenation is primarily catalyzed by the ActVA-5/ActVB system in ACT biosynthesis.

抄録全体を表示